The bacterial-like lactate shuttle components from heterotrophic Euglena gracilis

• Published in: Biochimica et biophysica acta Vol. 1709; no. 2; pp. 181 - 190
• Main Authors: Jasso-Chávez, Ricardo, García-Cano, Israel, Marín-Hernández, Álvaro, Mendoza-Cózatl, David, Rendón, Juan Luis, Moreno-Sánchez, Rafael
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 05.09.2005
• Subjects: Animals; Bacterial Proteins - chemistry; Energy Metabolism; Euglena gracilis - enzymology; Evolution, Molecular; Kinetics; Lactate Dehydrogenases - chemistry; Lactate Dehydrogenases - isolation & purification; Lactate Dehydrogenases - metabolism; Lactic Acid - chemistry; Lactic Acid - metabolism; Membrane-bound lactate dehydrogenase; Mitochondrion; Molecular Structure; NAD +-dependent lactate dehydrogenase; Mitochondrion; Energy metabolism; Membrane-bound lactate dehydrogenase; NAD +-dependent lactate dehydrogenase
• ISSN: 0005-2728; 0006-3002; 1879-2650
• DOI: 10.1016/j.bbabio.2005.07.007

The structural and kinetic analyses of the components of the lactate shuttle from heterotrophic Euglena gracilis were carried out. Mitochondrial membrane-bound, NAD +-independent d-lactate dehydrogenase ( d-iLDH) was purified by solubilization with CHAPS and heat treatment. The active enzyme was a 62-kDa monomer containing non-covalently bound FAD as cofactor. d-iLDH was specific for d-lactate and it was able to reduce quinones of different redox potential values. Oxalate and l-lactate were mixed-type inhibitors of d-iLDH. Mitochondrial l-iLDH also catalyzed the reduction of quinones, but it was inactivated during the extraction with detergents. Both l-iLDH and d-iLDH were inhibited by the specific flavoprotein-inhibitor diphenyleneiodonium, suggesting that l-iLDH was also a flavoprotein. Affinity chromatography revealed that the E. gracilis cytosolic fraction contained two types of NAD +-dependent LDH specific for the generation of d- and l-lactate ( d-nLDH and l-nLDH, respectively). These two enzymes were tetramers of 126–132 kDa and showed an ordered bi–bi kinetic mechanism. Kinetic properties were different in both enzymes. Pyruvate reduction by d-nLDH was inhibited by its two products; the d-lactate oxidation was 40-fold lower than forward reaction. l-lactate oxidation by l-nLDH was not detected, whereas pyruvate reduction was activated by fructose-1, 6-bisphosphate, K + or NH 4 +. Interestingly, membrane-bound l- and d-lactate dehydrogenases with quinone reductase activity have been only detected in bacteria, whereas the activity of soluble d-nLDH has been identified in bacteria and some yeast. Also, FBP-activated l-nLDH has been found solely in lactic bacteria. Based on their similar kinetic and structural characteristics, a possible common origin among bacterial and E. gracilis lactic dehydrogenase enzymes is discussed.